The goal of this project is to discover and optimize selective inhibitors of phosphoglycerate mutase (PGAM) and phosphoglycerate kinase (PGK) of trypanosomatid parasites (Trypanosoma brucei, T. cruzi and Leishmania species). These pathogens cause serious, often fatal diseases of humans such as sleeping sickness, Chagas' disease and kala-azar in tropical and subtropical countries primarily in Africa, Central and South America, and Asia where over 30 million people are infected, with approximately 130,000 deaths annually. Tragically, current drugs for their treatment are unsatisfactory because they are toxic and ineffective against some forms of the diseases, and resistance is becoming increasingly common. Glycolysis is essential in the infective stage of T. brucei (Tb) and therefore a promising drug target. Inhibitors of glycolytic enzymes such as PGAM and PGK may thus serve as lead compounds for the development of new drugs. PGAM is a particularly attractive target because the corresponding host enzyme is not homologous and has no features in common with the parasite enzyme. TbPGAM has been validated as a drug target by RNAi, and TbPGK by inhibitor studies. Detailed structural information is already available for both enzymes. The proposed research has as specific aims: (1) To develop and validate novel forward and reverse assays mounted on the NCGC high-throughput screening platform for trypanosomatid PGAM and PGK. The four assays will serve as both primary and orthogonal assays for confirmation of actives. (2) To use the four assays to screen the Molecular Library Small Molecule Repository (MLSMR) containing more than 300,000 small molecules. Unique structural features of trypanosomatid PGAM and PGK (compared with the corresponding human enzymes) will be exploited for the discovery of selective inhibitors of these enzymes initially from T. brucei and L. Mexicana. (3) To compare sets of actives in the forward and reverse directions. Preliminary assays on TbPGK have already shown the possibility to obtain inhibitors and activators of different chemotypes when assayed in the two directions. (4) To confirm the potency of these compounds in a panel of secondary hit validation assays and to test their specificity in kinetic and selectivity assays, and to further improve the potency of the most promising molecules thus obtained by structure-based methods, analogue synthesis and medicinal chemical principles. (5) To determine the mode of action of the most promising molecules by enzyme assays, protein crystallography and biophysical measurements. (6) To test compounds displaying the highest potency for their ability to inhibit growth of cultured trypanosomatid cells representing pathogenic stages of the parasites, as well as for lack of toxicity toward cultured human cells. PUBLIC HEALTH RELEVANCE: Sleeping sickness in sub-Saharan Africa, Chagas' disease in Central and South America, and kala-azar and related diseases in tropical and subtropical regions of the world cause severe public health and economic burdens on populations that are already caught in a tragic cycle of poverty, poor nutrition and disease. Millions of people worldwide are infected by these potentially fatal diseases and hundreds of millions are at risk. Existing treatments have developed little in the past 50 years, and suffer from toxicity, inefficiency and resistance; the goal of this project is to develop lead drugs that will be suitable for entry into pre-clinical trials.